This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. SfiI is a teterameric endonuclease that requires the binding of two DNA duplexes for successful activation and concerted cleavage of all four strands. We have solved the structure of the native form of the enzyme in complex with DNA and the structure indicates that there is a cross-talk between monomers that is responsible for allosteric activation of SfiI. We now have a mutant (Y68F)that is still tetrameric but preferentially binds and cleaves one ds DNA. We have crystals of this mutant that diffracts to 4-4.5 A at the home source. We are looking to collect high resolution data to uncover the role of Y68 in the allosteric activation of SfiI.